JP2001305105A - Flat type gel electrophoretic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a flat type gel electrophoretic apparatus which permits integration of all processes from the generation of a DNA sample to be analyzed to the electrophoresis. SOLUTION: In the flat type gel electrophoretic apparatus in which a fluorescence labelled DNA sample is separated with a gel eletrolyte layer of an electrophoretic plate placed horizontally to receive fluorescence released from the separated individual bases by a line sensor for detecting fluorescence, the electrophoretic plate comprises upper and lower members and the upper member has a through hole for pouring a sample, a through hole for pouring an upper buffer liquid, a through hole for pouring a lower buffer liquid, a gel eletrolyte injection port near the through hole for pouring the upper buffer liquid and a gel electrolyte ejection port near the through hole for pouring the lower buffer liquid. A sunk part for a sample generating reaction and a groove with a specified length for housing a gel electrolyte are cut into one plane of the lower member at positions corresponding to the through hole for pouring the sample of the upper member. Then, the upper member and the lower member are laminated integrally to form the electrophoretic plate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gel electrophoresis apparatus. More specifically, the present invention relates to a flat-plate gel electrophoresis apparatus having a DNA sample preparation function and capable of performing all steps from sample preparation to electrophoresis.

[0002]

2. Description of the Related Art Gel electrophoresis is widely used as a method for determining a base sequence of DNA or the like.

Conventionally, when performing electrophoresis, a sample is labeled with a radioisotope and analyzed, but this method has a drawback that it requires time and effort. In addition, maximum safety and control are always required from the viewpoint of radioactivity management, and analysis cannot be performed unless in a special facility. For this reason,
Recently, a method of labeling a sample with a phosphor has been studied.

In the method using light, a fluorescently labeled DN is used.
Run the A fragment in the gel. For example, DNAs of various lengths whose terminal base species are known by an enzymatic reaction (dideoxy method) using a DNA strand whose sequence is to be determined as a template
NAs are replicated and these are labeled with fluorophores. That is, an adenine (A) fragment group, a cytosine (C) fragment group, a guanine (G) fragment group, and a thymine (T) fragment group labeled with a fluorescent substance are obtained. These fragment groups are mixed, injected into separate electrophoresis lane grooves of an electrophoresis gel, and a voltage is applied. Since DNA is a chain polymer polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Shorter (small molecular weight) DNA strands move faster and longer (larger molecular weight) DNA chains move more slowly, so that DNA can be fractionated by molecular weight.

As a method of electrophoresing a fluorescently labeled DNA fragment in a gel, a plate-type (or also called “flat-bed”) gel electrophoresis apparatus as shown in FIG. 2 is used. For example, a gel electrolyte layer 102 such as agarose or polyacrylamide is formed on the upper surface of a transparent glass or quartz substrate 100, and this gel electrolyte layer 10
The excitation light source 104 is arranged on the upper part of the
4 and a light receiving optical system 106 on the opposite side of the substrate 102.
Is arranged, and a fluorescence detector 108 such as a linear line sensor is arranged below the optical system 106. Fluorescence detector 1
A scanning aperture slit 110 for defining a fluorescence incident area at 08 is located immediately before the fluorescence detector 108. An optical filter 112 is also provided below the substrate. This filter has uniform optical characteristics, and in an actual analysis, first receives excitation light from the light source 104 and generates a reference value for calibration.

FIG. 3 is a plan view of the device shown in FIG. When a voltage is applied, the analyzed DNA sample 114 spotted at the migration start point of each migration path of the gel electrolyte layer 102 moves in the gel electrolyte layer 102 at a speed inversely proportional to the molecular weight, and is separated for each base 116. Is done. Fluorescence from each separated base passes through the scanning aperture slit 110 and is received and detected by each of the pixels 108a to 108n of the linear line sensor 108.

However, in the conventional plate-type gel electrophoresis apparatus, due to the problem of the size of the electrophoresis plate and the process of generating a sample to be analyzed, the system from the sample generation to the electrophoresis is fully automated, and the configuration thereof becomes huge. Therefore, it has hardly been put to practical use until now. For this reason, in the conventional plate-type gel electrophoresis apparatus, the DNA sample to be analyzed has to be prepared in another place, and the prepared sample has to be manually loaded on the gel electrolyte layer using a tool such as a capillary. However, the throughput of the analysis operation could not be improved.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a plate-type gel electrophoresis apparatus capable of consistently performing the steps from preparation of a DNA sample to be analyzed to electrophoresis.

[0009]

The object of the present invention is to provide a through hole for injecting a sample, a through hole for injecting an upper buffer solution, a through hole for injecting a lower buffer solution, and a vicinity of the through hole for injecting an upper buffer solution. A gel inlet, an upper member having a gel outlet near the through hole for lower buffer solution injection, a concave portion for sample preparation reaction on one flat surface, and a predetermined portion for containing a gel electrolyte. The problem is solved by a flat-plate gel electrophoresis apparatus characterized by using a migration plate comprising a lower member having a groove having a length.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a flat plate gel electrophoresis apparatus of the present invention will be described in detail with reference to the drawings.
FIG. 1 is a schematic exploded perspective view showing an example of the configuration of the flat plate gel electrophoresis apparatus of the present invention. The electrophoresis plate in the flat gel electrophoresis apparatus of the present invention basically includes an upper member 1 and a lower member 3. The upper member 1 is provided with a through hole 5 for sample injection, a through hole 7 for upper buffer solution injection, and a through hole 9 for lower buffer solution injection. Further, a gel inlet 11 is provided near the upper buffer solution injecting hole 7 of the upper member 1 and a gel outlet 13 is provided near the lower buffer solution injecting hole 9. A gel injection syringe 15 is connected to the gel injection port 11. Switching valves 17 and 19 for switching between the injection (and discharge) of the gel and the injection (and discharge) of the buffer solution are provided. The gel injection syringe 15 is driven to inject (and discharge) the gel.
In this case, the switching valves 17 and 19 are pulled out, and the buffer liquid inlets 7 and 9 are closed. When performing electrophoresis, push in the switching valves 17 and 19 to set the gel injection port 1
1 and the gel outlet 13 are closed, and the buffer solution inlets 7 and 9 are opened.

On one plane of the lower member 3, a concave portion 21 for receiving an injected sample or the like is provided at a position corresponding to the position of the sample injection through hole 5 of the upper member 1.
Is engraved. A sample generation reaction section 23 communicating with the recess 21 is provided. The sample and the reagents can be directly injected into the sample generation and reaction unit 23 from the sample injection through hole 5 without providing the recess 21. The volume of the sample generation reaction section 23 is 20 μl
It is preferably in the range of １００100 μl. 20 μl
If it is less than 10, it is difficult to accommodate the reagents necessary for the PCR method and the sequence reaction, and the yield of the product is too small to hinder the analysis. If it exceeds 100 μl, it will be too large and useless. A sample loading section 25 is engraved on the same axis as the sample generation reaction section 23 and adjacent to the sample generation reaction section 23. The position of the sample loading section 25 corresponds to the position of the upper buffer solution injection through-hole 7. The electrophoresis section 27 extends from the sample loading section 25 to the electrophoresis end section 27. The position of the electrophoresis ending section 27 corresponds to the position of the lower buffer solution injection through-hole 9. A heating means 37 is provided below the sample generation reaction section 23, and can heat the sample and the reaction reagents injected into the sample generation reaction section 23 to a predetermined temperature.

The recess 21, the sample generation reaction unit 23, the sample loading unit 25, the electrophoresis unit 27, and the electrophoresis termination unit 27 on the plane of the lower member 3 are formed by a conventional etching technique such as photolithography using a photoresist. Can be engraved. As the material of the upper member 1 and the lower member 3, any material such as light-transmitting glass, quartz, or plastic can be used.

In the plate-type gel electrophoresis, as the thickness of the gel electrolyte layer is reduced, the band separation ability is improved. Therefore, when reading the same base pair length, if the gel electrolyte layer is made thinner, the whole apparatus can be downsized as compared with the conventional electrophoresis plate size. Further, as shown in the drawing, by making the electrophoresis section 27 meander in a substantially S-shape, the entire apparatus can be significantly reduced in size as compared with the case where the electrophoresis section 27 is engraved in a straight line. it can.

The detection board 35 on which the optical filter 31 and the fluorescence detection line sensor 33 are mounted can scan the electrophoresis section 27 by a known and commonly used means. Although not shown, any light source such as an ultraviolet light source or a laser light source can be used as the excitation light source, and such a light source can be disposed above the upper member 1 or the detection substrate 35 can be used. It can also be located on the same side as.

FIG. 1 shows two migration paths, but the number of migration paths to be provided may be one, or may be three or more. In accordance with the number of migration paths to be provided, the sample injection through-hole 5, the upper buffer solution injection through-hole 7, the lower buffer solution injection through-hole 9, the sample generation reaction unit 23, and the like may be provided.

Next, the preparation of a DNA sample by the plate-type gel electrophoresis apparatus of the present invention will be described. The DNA sample is generated in a sample generation reaction unit 23 engraved on the plane of the lower member 3. Basically, a DNA sample is prepared by a known PCR method and a sequencing reaction. P
The CR method is a polymerase chain reaction method comprising amplifying a specific region several hundred thousand times by repeating a DNA synthesis reaction between two types of primers and a DNA synthase sandwiching the specific DNA region. , Developed by Cetus of the United States in 1985. This method
Since then, the method has been improved to a method using the thermostable enzyme Taq polymerase, which has been established as an efficient PCR method, and has reached the present. The principle of the PCR method is described in, for example, Experimental Medicine, Vol. 7, No. Pages 2, 14 to 18 (19
89). This PCR method basically denatures a double-stranded DNA to which a primer is bound into a single strand by heat denaturation, anneals the primer,
It consists of a series of cycles consisting of extension with Taq polymerase, and the amplification of a desired DNA fragment by repeating this cycle several tens of times.

PCR amplification can be performed using a kit commercially available from Perkin-Elmer, USA. The reaction mixture kit contains 3 μl of template DNA (for example,
Human genome about 200ng), 10μ PCR buffer 3μ
1, 3 μl of dNTP (2.5 mmol), Primer 1
(10 μmol) 3 μl, Primer 2 (10 μmol) 3 μl, Ex Taq 0.2 μl and H ₂ O 1
It is a 30 μl total volume solution consisting of 5 μl. Other reaction mixture kits can also be used. This is injected into the sample generation reaction section 23 through the sample injection through hole 5, and the heating means 37 is driven to heat the injected reagents at 96 ° C. for 3 minutes, and then at 96 ° C. for 30 seconds and 68 ° C. And heating for 3 minutes as one cycle, and repeat for 25 to 30 cycles.
1 μl of the obtained PCR amplification product is collected, and the amplification state is checked using a mupid. If amplification is insufficient, the heating cycle is repeated. If sufficiently amplified, an enzyme is added to decompose unreacted substances in the PCR amplification product. As enzymes, Alkaline Phosphatase Shrimp (APS) for removing unreacted dNTPs and Exonuklease I (Exol) for removing unreacted primers are used. Dilute each enzyme 20-fold with TE and add 2 μl each. Then, heat at 37 ° C for 15 minutes, and further heat at 80 ° C.
For 15 minutes to decompose unreacted substances. Thereafter, the obtained purified product is subjected to a sequence reaction.

As the sequence reaction, a cycle sequence reaction can be used. This reaction is one of the sequence reaction methods by the dideoxy method,
Using the R method, four types of deoxynucleotide triphosphates (dNTP, N are A, C, G, T) are each added to one type of dideoxynucleotide triphosphate (ddNTP, N is A,
(C, G, T) using a mixed reagent, adding one kind of primer, and performing a thermal cycle reaction. For example, in the sample generation reaction part 23, 4.5 μl of 5 × bent enzyme buffer, analysis DNA to be amplified by the PCR method
The sample, 1.8 picomoles of primer, 2 units of vent enzyme and H ₂ O are added to make a total volume of 18 μl. In addition, d
/ DdATP, d / ddCTP, d / ddGTP or d
Then, 1.0 μl of / ddTTP is added, and the reaction is performed by the PCR method. A heat cycle of 15 seconds / 95 ° C.-45 seconds / 64 ° C.-60 seconds / 70 ° C. is repeated 30 times, and then 2 μm
Add 1 formaldehyde dye. Since the temperature during annealing depends on the number and length of bases of the primer,
It is preferable to match the optimum value each time.

Fluorescent label D obtained by sequence reaction
The NA sample is taken out of the sample generation reaction unit 23 by a known and commonly used means such as a capillary or a micropipette, and is loaded into the sample loading unit 25 through the upper buffer solution injection through hole 7. Thereafter, a buffer solution is poured into each of the upper buffer solution injection through-hole 7 and the lower buffer solution injection through-hole 9, and a voltage is applied between the sample loading section 25 and the electrophoresis ending section 27 to cause electrophoresis. Let it. The voltage can be applied by inserting a power supply terminal (not shown) into the upper buffer solution injection through-hole 7 and the lower buffer solution injection through-hole 9. Fluorescence emitted from each base separated by electrophoresis is detected by a fluorescence detection line sensor 33, similarly to a conventional flat plate gel electrophoresis apparatus.

[0020]

As described above, according to the present invention,
A miniaturized plate-type gel electrophoresis apparatus capable of performing from the preparation of an analyte DNA sample to the electrophoresis consistently is obtained. Particularly, in the sample generation process, the conventional P
By performing the purification process after CR amplification using enzymes instead of using a centrifuge, all the steps of sample generation can be performed in the same reaction vessel, greatly reducing the time required for sample generation work. Is done. Moreover, after sample generation,
Since the DNA sequencing analysis can be started immediately by electrophoresis, the total working time from sample generation to sequencing is also significantly reduced.

[Brief description of the drawings]

FIG. 1 is a schematic exploded perspective view showing an example of the configuration of a flat panel gel electrophoresis apparatus of the present invention.

FIG. 2 is a schematic diagram showing an example of the configuration of a conventional flat plate gel electrophoresis apparatus.

FIG. 3 is a schematic plan view showing an electrophoresis state in the flat plate gel electrophoresis apparatus shown in FIG.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Upper member 3 Lower member 5 Sample injection through hole 7 Upper buffer liquid injection through hole 9 Lower buffer liquid injection through hole 11 Gel electrolyte injection port 13 Gel electrolyte discharge port 15 Gel electrolyte injection syringe 17, 19 Switching valve 21 Recessed part 23 Sample generation reaction part 25 Sample loading part 27 Electrophoresis termination part 29 Electrophoresis part 31 Optical filter 33 Fluorescence detection line sensor 35 Substrate

 ────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Shinichi Takagi 3-16-3 Higashi, Shibuya-ku, Tokyo F-term in Hitachi Electronics Engineering Co., Ltd. 2G043 AA04 BA16 CA03 DA05 EA01 EA19 FA01 GA04 GB01 JA03 KA03 KA09 LA03

Claims (4)

[Claims] 1. A flat plate type comprising separating a fluorescence-labeled DNA sample at a gel electrolyte layer of a horizontally mounted electrophoresis plate and receiving fluorescence emitted from each separated base by a fluorescence detection line sensor. In the gel electrophoresis apparatus, the electrophoresis plate includes an upper member and a lower member, wherein the upper member has a through hole for injecting a sample, a through hole for injecting an upper buffer solution, and a through hole for injecting a lower buffer solution. A hole, a gel electrolyte inlet near the upper buffer solution injection through-hole, and a gel electrolyte outlet near the lower buffer solution injection through-hole, on one plane of the lower member, A concave portion for sample generation reaction is provided at a position corresponding to the through hole for sample injection of the upper member, and a groove of a predetermined length for accommodating a gel electrolyte is engraved, and the upper member and the lower member are Flat plate type gel electrophoresis apparatus, characterized in that the electrophoresis plate is constituted by integrating roots aligned. 2. A groove having a predetermined length for accommodating the gel electrolyte includes a sample loading portion corresponding to a position of an upper buffer solution injecting hole of the upper member, an electrophoresis portion, and the upper member. And a continuous groove comprising an electrophoresis ending portion corresponding to the position of the through-hole for injecting the lower buffer solution, wherein the electrophoresis portion is formed in a substantially S-shape. The flat plate gel electrophoresis apparatus according to the above. 3. A switching valve for switching between the through hole for injecting the upper buffer solution and the gel electrolyte inlet, and a switching valve for switching between the through hole for injecting the lower buffer solution and the gel electrolyte outlet. The flat-plate gel electrophoresis apparatus according to claim 1, further comprising a switching valve of (1). 4. The flat panel gel electrophoresis apparatus according to claim 1, wherein a heating means is further provided on a lower surface of the concave portion for sample generation reaction of the lower member.